IMMOBILIZED METAL AFFINITY MEMBRANE ADSORBERS AS STATIONARY PHASES FOR METAL INTERACTION PROTEIN SEPARATION

Novel immobilized metal affinity membrane adsorbers (IMA-MA) were stud ied for potential use as stationary phases for protein separation. Pro tein adsorption on IMA-MA loaded with Cu(II), Ni(II), Zn(II) and Co(II ) ions was compared as a function of the flow-rate and the ionic stren gth of the elution buffer. To exclude the possibility of mixed-mode in teraction in the experiments, the binding of proteins similar in terms of hydrophobicity, isoelectric point, size and mass-to-charge ratio b ut differing in their number of surface histidine residues was investi gated. Matrix-assisted laser desorption/ionization mass spectrometry w as used to distinguish between these proteins in the eluted fractions. Salt concentration of at least 0.5 M NaCl and flow-rates below 2 ml m in-1 were found suitable to ensure an adsorption mechanism based on af finity interaction between the proteins and the chelated metal ions. I n an application study, the IMA-MA and conventional chelating Sepharos e fast flow columns were compared for the isolation of a recombinant f usion protein (EcoR V), which carried a polyhistidine sequence (HIS6-t ag) at the N-terminus.